cancers

Review
The Exciting New Field of HER2-Low Breast Cancer Treatment
Daniel Eiger 1 , Elisa Agostinetto 1,2 , Rita Saúde-Conde 1,3 and Evandro de Azambuja 1, *

1 Academic Promoting Team, Institut Jules Bordet, L’Universite Libre de Bruxelles (U.L.B.),
1000 Brussels, Belgium; danieleiger@gmail.com (D.E.); elisa.agostinetto@bordet.be (E.A.);
rconde@ipolisboa.min-saude.pt (R.S.-C.)
2 Medical Oncology and Haematology Unit, Humanitas Cancer Center, Humanitas Clinical and Research
Center—IRCCS, Rozzano, 20089 Milan, Italy
3 Medical Oncology Department, Instituto Português de Oncologia de Lisboa Francisco Gentil,
1099-023 Lisbon, Portugal
* Correspondence: evandro.azambuja@bordet.be

Simple Summary: Breast cancer can express, at varied levels, a protein named HER2, commonly
responsible for making it grow and send distant metastases. In the past, patients affected by the so
called HER2-positive breast cancer had lower probabilities of cure and survival, though with the
advent of drugs that target HER2, three decades ago, their prognosis has greatly improved. So far,
only patients with strong HER2 expression on their tumour can be treated with these benefitial drugs,
like trastuzumab, though recently stronger drugs have also been shown capable of eliminating breast
cancer cells with lower levels of HER2 expression (HER2-low). Sooner or later, these new drugs,
like trastuzumab-deruxtecan, may be available for treating such patients. Therefore, the aim of this
narrative review of the literature is to provide an outline of what is going on on this specific field of
research, and what could be expected in the future in the clinic.






Abstract: Since human epidermal growth factor receptor-2 (HER2) characterization, going through
Citation: Eiger, D.; Agostinetto, E.; clinical research and regulatory approval of HER2-targeted therapies, much has elapsed and is still
Saúde-Conde, R.; de Azambuja, E. unfolding. Hitherto, only breast cancer (BC) patients with HER2 immunohistochemistry 3+ or with
The Exciting New Field of HER2-Low HER2 gene fluorescence in-situ hybridization (FISH) amplification (a.k.a., HER2-positive BC) have
Breast Cancer Treatment. Cancers benefited from anti-HER2 agents. In recent years, however, much of the research effort has been
2021, 13, 1015. https://doi.org/
expanded, with positive outcomes being reached for formerly known HER2-negative BC that yet
10.3390/cancers13051015
express HER2 to some degree (HER2 immunohistochemistry 1+ or 2+, but FISH negative) and are
currently being classified as HER2-low BC for the purpose of trial enrollment. In this sense, our aim
Academic Editor: David Wong
is to review the body of evidence of HER2-low BC that led to the study of first-generation anti-HER2
Received: 3 February 2021 agents, like trastuzumab, and how they have failed to achieve any clinical applicability in this setting.
Accepted: 24 February 2021 In addition, we review new data that is leading to the growing success of the new generation of
Published: 1 March 2021 drugs, especially the promising HER2-directed antibody–drug conjugates. A narrative review is
also performed regarding the rationale behind the consolidated and ongoing clinical trials studying
Publisher’s Note: MDPI stays neutral anti-HER2 agents in combination with unrelated agents, such as immunotherapy, endocrine therapy,
with regard to jurisdictional claims in and CDK4/6 inhibitors. Hopefully, all this ongoing research effort will be able to extend the survival
published maps and institutional affil- benefits seen with anti-HER2 agents in HER2-positive disease, at least to some degree, to the greater
iations. proportion of patients with HER2-low BC.

Keywords: HER2-low breast cancer; trastuzumab; antibody–drug conjugates; trastuzumab–deruxtecan;

trastuzumab–duocarmazine; zenocutuzumab
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and 1. Introduction
conditions of the Creative Commons
Drugs targeting the human epidermal growth factor receptor-2 (HER2) have revolu-
Attribution (CC BY) license (https://
tionized the treatment landscape of HER2-positive breast cancer (BC) patients, creating
creativecommons.org/licenses/by/
a new standard of remarkable survival outcomes for once a BC subtype with gloomy
4.0/).

Cancers 2021, 13, 1015. https://doi.org/10.3390/cancers13051015 https://www.mdpi.com/journal/cancers

Cancers 2021, 13, 1015 2 of 18

perspectives [1–5]. Early trials testing trastuzumab, the first anti-HER2 drug ever ap-
proved, demonstrated that tumor responses were restricted to patients whose tumors
stained 3+ for HER2 on immunohistochemistry (IHC) or stained 2+ but had HER2 gene
amplification (≥2 copies) on fluorescence in-situ hybridization (FISH) [6–8]. These early
observations have established the standard to which subsequent trials and international
guidelines would test and recommend anti-HER2 therapies, respectively [9–12]. While
HER2-positive BC comprises only around 20% of newly diagnosed cases, a greater propor-
tion of patients (≈40–50%) have BC categorized as HER2-low, i.e., IHC of 1+ or 2+ but FISH
negative [13–15]. Nonetheless, HER2-low BC is considered altogether with those with 0+
at IHC as HER2-negative, for the purpose of current treatment decisions (i.e., non-eligible
for anti-HER2 therapies) [14–16].
Patients with HER2-low BC spam a heterogeneous group, immunohistochemically
comprised of a majority of hormone receptor (HR) positive tumors (65–83%), while the
rest has HR-negative tumors [17,18]. As such, HR-positive, HER2-low BC has a distinct
molecular profile than HR-negative, HER2-low BC: while the first is enriched with luminal
subtypes, the latter demonstrates a predominance of the basal-like subtype, underlining
major genetic, clinicopathological, and prognostic differences within the group [17].
Though treated in the same manner as patients with HER2 IHC 0+ BC, patients with
HER2-low breast cancer may portray a clinical picture closer to that of HER2-positive
BC patients: in a large prospective series, patients with HER2 IHC 2+, FISH-negative BC
tended to present with larger tumor size, higher histopathological grade, higher Ki67, and
more frequently with axillary nodal involvement [19]. Likewise, disease-free survival of
these patients was inferior to that of patients with HER2 0+ and, after the introduction
of adjuvant trastuzumab, inferior to that of those with HER2-positive BC. These findings
were, to different degrees, replicated in additional series and further defined a subgroup
of patients within the HER2-negative space with tumors with HER2 expression levels
sufficient enough to exert some oncogenic effect, ultimately putting them in need of
additional therapies [20,21].
The targeted treatment options for patients with HER2-low BC will either end upon
failure of sequenced endocrine therapies (ET; tamoxifen, aromatase inhibitors or fulvestrant
with or without cyclin-dependent kinase 4 and 6 inhibitors or everolimus or alpelisib),
in case of HR positivity or are restricted to a subset of those with HR negativity (namely
immunotherapy for patients whose tumors are positive for programmed death-ligand
1 (PD-L1) receptor expression or poly(ADP-ribose) polymerase inhibitors for those with
germline BRCA mutations) [11,12]. Reliance on traditional cytotoxic chemotherapies thus
ensues, a strategy with known constraints, namely dose-limiting, cumulative toxicities
and limited survival gains [11,22]. Extending the survival benefits seen with anti-HER2
agents to this expressive parcel of patients is, therefore, an attractive venue, given the poor
outcomes of triple-negative BC (TNBC) patients and of endocrine-resistant, HR-positive
BC patients [11,23].
In the past, trastuzumab has failed to improve the outcomes of patients with HER2-low BC,
and the concept of anti-HER2 agents in this setting was put on hold [18]. Fortunately, this treat-
ment paradigm has recently been re-challenged in light of the promising efficacy results seen with
novel and more potent anti-HER2 agents in HER2-low metastatic BC [24–26]. It is, therefore, the
objective of this article to review the previous body of data and upcoming evidence for the
new wave of treatments that may revolutionize the care of HER2-low BC patients. From
trastuzumab to trastuzumab–emtansine, moving to trastuzumab–deruxtecan and combina-
tions with immunotherapy, endocrine therapies, cyclin-dependent kinase inhibitors, among
others, preclinical, clinical, and safety data supporting further testing of anti-HER2 drugs
for the treatment of HER2-low BC patients are reviewed and put into perspective below.

2. Rationale for Targeting HER2-Low BC with Anti-HER2 Agents

HER2-low BC cell lines express a considerable quantity of targetable HER2 [27]. In
this regard, though it mechanistically made sense that anti-HER2 agents could have clinical
Cancers 2021, 13, 1015 3 of 18

applications beyond HER2-positive BC, this was never achieved in the first two decades of
experience with HER2-targeted monoclonal antibodies (trastuzumab and pertuzumab),
anti-HER2 vaccine (nelipepimut-S), and the first anti-HER2 antibody-drug conjugate (ADC)
(trastuzumab–emtansine) [18,28–30].

2.1. Trastuzumab
Trastuzumab was once hypothesized to work in early-stage BC with minimum levels
of HER2 expression: other than blocking the HER2 growth signaling pathway, trastuzumab
also causes antibody-dependent cellular cytotoxicity (ADCC). ADDC could play a bigger
role in the micrometastatic (adjuvant) setting, where the bulky of disease and tumor-
induced immunosuppression are lower than in the metastatic setting; thereby, the level of
HER2 expression in the tumor is less important for trastuzumab activity [19,20]. Moreover,
in the micrometastatic setting, mice xenograft models of HER2-negative luminal BC have
been shown to have their implants’ growth driven by HER2 upregulation in the bone,
a common site of metastatic seeding. When the mice were given trastuzumab shortly
after tumor implantation, despite the original absence of HER2 overexpression in the
model, tumor growth could be halted, forming the basis to postulate an adjuvant effect of
trastuzumab on HER2-negative disease [31].
In the clinical setting, post hoc retrospective analysis of two phase 3 trials further
corroborated this hypothesis. In NSABP B-31 and NCCTG N9831, comparing adjuvant
chemotherapy with trastuzumab vs. chemotherapy alone, patients were initially eligible
based on local laboratory HER2 assessment. Discordant cases, where final tumor results
for HER2 were actually negative upon central pathology review, were thus included [32].
In B-31, 9.7% of patients were centrally-assessed as HER2-negative, and yet they derived a
beneficial effect from trastuzumab (relative risk for disease-free survival (DFS), 0.34; 95%
CI, 0.14 to 0.80), similarly to HER2-positive patients [33]. Moreover, HER2 messenger RNA
levels were consistently lower in the HER2-negative tumors compared to the HER2-positive
tumors, providing further evidence that HER2-negativity at the central pathology review
was not a false-negative result at the transcriptomic level and yet patients were benefiting
from trastuzumab. In N9831, among 5.5% centrally assessed HER2-negative BC, a trend
towards benefit with trastuzumab was found (hazard ratio (HR) for DFS, 0.51; 95% CI,
0.21–1.23) [34].
Despite these early positive signals, NSABP B-47, a large randomized phase 3 trial that
enrolled 3270 patients with HER2-low BC to adjuvant trastuzumab plus chemotherapy vs.
chemotherapy alone, failed to prove any beneficial effect of trastuzumab (HR for invasive
DFS, 0.98; 95% CI, 0.76 to 1.25) (Table 1) [18]. In subgroup analyses, even patients with a
higher degree of HER2 expression (IHC 2+) did not benefit from trastuzumab, similarly to
patients with HER2 IHC 1+ tumors.

Table 1. Key clinical efficacy data of anti-human epidermal growth factor receptor-2 (HER2) agents in HER2-low BC.

Author Study Design Study Population N Treatment Main Efficacy Results

Single anti-HER2 agents
5-year iDFS: 89.8% vs.
High-risk early 89.2%, HR 0.98; 95% CI,
Phase 3, Adjuvant ChT
Fehrenbacher et al. BC-negative for 0.76–1.25; p = 0.85/
randomized 3270 with or without
NSABP B-47 [18] HER2 by FISH and OS: 94.8% vs. 96.3%, HR
(1:1) trial trastuzumab
with IHC 1+ or 2+ 1.33; 95% CI, 0.90–1.95;
p = 0.15
CBR (CR + PR + SD at 24
weeks): 9.8% in 420 mg
Phase 2, Pertuzumab (420 q3w arm vs. 5.4% in
HER2-low
Gianni L et al. [28] randomized 78 mg q3w vs. 1050 1050 mg q3w arm
metastatic BC
(1:1) trial mg q3w) Median time to
progression: 6.1 weeks
(both arms)
Cancers 2021, 13, 1015 4 of 18

Table 1. Cont.

Author Study Design Study Population N Treatment Main Efficacy Results

ORR: 4.8% (95% CI,
HER2-positive 1.0–21.8%) vs. 33.8%
metastatic BC (95% CI, 23.2–44.9%)
Phase 2, single-arm 112 pts (21 Trastuzumab
Burris et al. [35] (including Median PFS: 2.6 mo
trial HER2-low) emtansine (T-DM1)
HER2-low BC after (95% CI, 1.4–3.9 mo) vs.
central assessment) 8.2 mo (95% CI, 4.4 mo
to NE)
HER2-positive ORR: 20% (95% CI,
metastatic BC 5.7–44.9) vs. 41.3% (95%
Phase 2, single-arm (including 110 pts (15 Trastuzumab CI 30.4–52.8)
Krop et al. [36]
study HER2-low BC after HER2-low) emtansine (T-DM1) Median PFS: 2.8 mo
retrospective (95% CI 1.3-NE) vs. 7.3
re-evaluation) (95% CI, 4.6–12.3)
ORR: 37% (95% CI,
Phase 1, HER2-low BC Trastuzumab–
24.3–51.3%)
Modi et al. [24] dose-expansion refractory to 54 deruxtecan
Median DoR: 10.4 mo
study standard therapies (T-DXd) (DS8201a)
(95% CI, 8.8 mo-NE)
ORR: 28% (95% CI,
Advanced BC,
13.8–46.8%) in HR+
Phase 1 gastric, urothelial, 146 (47 Trastuzumab
HER2-low BC, 40% (95%
Banerji et al. [25] dose-expansion or endometrial HER2-low duocarmazine
CI, 16.3–67.6%) in HR-
study cancer with at least BC) (SYD985)
HER2-low BC
HER2 IHC 1+

Combination therapies
24-month DFS rate:
89.9% in the vaccine arm
Node-positive (or vs. 83.8% in the control
negative if arm (HR = 0.62; 95% CI
Phase II, Nelipepimut-S +
HR-negative) = 0.31–1.25; p = 0.18);
Hickerson et al. randomized (1:1), trastuzumab vs.
HER2-low BC 275 24-month DFS rate in
[29] blinded, placebo + GM-CSF
patients after the subgroup of TNBC:
placebo-controlled + trastuzumab
standard adjuvant 92.6% vs. 70.2%,
therapy respectively (HR = 0.26;
95% CI = 0.08–0.81;
p = 0.013)
Baseline mean Ki67:
32.4%
Cohort C:
Trastuzumab + Mean Ki67 at week 2:
Phase II, HR-
pertuzumab + 2.6% (mean a change of
Gianni et al. [37] multicenter, positive/HER2- 23
fulvestrant + −29.5; p < 0.001)
multicohort trial low early
palbociclib Mean Ki67 at surgery:
BC
7.5% (mean change of
−19.3; p < 0.001)
ER+/HER2-low
CBR (CR + PR + SD at 24
metastatic BC Zenocutuzumab
Pistilli et al. [26] Phase 2 study 50 weeks): 16.7% (90% CI
refractory to (MCLA-128) + ET
8.6–28.1)
ET/CDK4/6i
Confirmed ORR by
Cohort 2: Trastuzumab– independent central
2-part, phase 1b
Hamilton et al. [38] HER2-low BC after 16 deruxtecan + review: 38% (95% CI,
study
standard therapy nivolumab 15–65);
DoR not evaluable
Legends: BC: breast cancer; CBR: clinical benefit rate; CDK 4/6i: cyclin-dependent kinase 4/6 inhibitors; CI: confidence interval; CR:
complete response; DoR: duration of response; ER: estrogen receptor; ET: endocrine therapy; GM-CSF: granulocyte macrophage-colony
stimulating factor; HR: hazard ratio; iDFS: invasive disease-free survival; IHC: immunohistochemistry; NE: not evaluable; ORR: overall
response rate; NSCLC: non-small cell lung cancer; PFS: progression-free survival; PR: partial response; RFI: relapse-free interval; SD:
stable disease.
Cancers 2021, 13, 1015 5 of 18

2.2. Pertuzumab
Pertuzumab is another monoclonal antibody targeted against HER2, which prevents
its homodimerization and also heterodimerization with other activating HER family part-
ners, further blocking downstream growth signaling activation [39]. Unlike trastuzumab,
pertuzumab is capable of inhibiting tumor growth of xenograft models even in the absence
of HER2 overexpression [40]. Nonetheless, pertuzumab alone, given for previously treated
HER2-negative or low BC patients, yielded disappointing tumor responses in a phase II
trial (Table 1) [28].

2.3. Nelipepimut-S
E75 is a HER2-derived peptide capable of stimulating CD8+ T cytotoxic lymphocytes
to recognize and eliminate HER2-expressing cancer cells [41]. Combined with an im-
munoadjuvant (granulocyte macrophage-colony stimulating factor (GM-CSF)), the HER2-
targeting vaccine nelipepimut-S has been shown to induce E75-specific CD8+ T-cells expan-
sion, which is even greater in patients with HER2-low BC [42]. Given with trastuzumab,
the expansion of E75-specific CD8+ T-cells is amplified, underlying a synergism for the
combination [43].
In this sense, an early immunotherapeutic approach tested in clinical trials of HER2-
low BC patients was nelipepimut-S combined with trastuzumab. It was evaluated after
standard adjuvant therapy for patients with HR-negative/HER2 IHC 1+ or 2+ (FISH-
negative), node-negative BC or node-positive BC regardless of HR status [29]. Patients
were randomly assigned to receive trastuzumab once every 3 weeks for 1 year and placebo
with GM-CSF (control arm; n = 139) or nelipepimut-S (experimental arm; n = 136). In the
intention-to-treat analysis, no statistical difference was observed for the primary endpoint
(24-month DSF-rate of 89.9% in the vaccine arm vs. 83.8% in the control arm (HR = 0.62; 95%
CI = 0.31–1.25)), albeit in the subgroup of patients with HR-negative BC, nelipepimut-S was
able to significantly improve it (Table 1). Still, nelipepimut-S development for HER2-low
BC did not move forward.

2.4. Trastuzumab-Emtansine
Trastuzumab–emtansine (T-DM1) is a 2nd generation ADC composed of the HER2-
targeting vehicle trastuzumab, bound via a non-cleavable thioether linker to the potent
anti-tubulin, maytansine derivative DM1, with a drug–antibody ratio of 3.5:1. Its antitumor
properties reside not only on the blockade of the HER2 signaling pathway and ADCC
induction by trastuzumab but also on the internalization of the cytotoxic moiety by HER2
expressing cells, therefore, having a more potent cytotoxic effect within tumor cells instead
of on healthy tissues (i.e., a better therapeutic index than traditional cytotoxic drugs) [44].
Unlike trastuzumab, T-DM1 was never prospectively tested in HER2-low BC. Nonethe-
less, in two phases 2 trials testing the efficacy and safety of T-DM1 in HER2-positive
metastatic BC patients previously treated with at least trastuzumab, retrospective, ex-
ploratory analyses according to central laboratory assessment of HER2 status found poor
clinical activity of T-DM1 among patients with HER2-normal BC compared to patients
with HER2-positive BC (Table 1) [35,36].
T-DM1 was, however, prospectively tested in the analogous setting of HER2-positive
but heterogeneous BC [30]. Genetic heterogeneity is present in a significant proportion
of BCs that would otherwise be classified as HER2 FISH-negative [45]. In a phase 2
study of neoadjuvant T-DM1 plus pertuzumab, HER2 heterogeneity was found in 10% of
cases (16 out of 157 enrolled patients). No patients with HER2 heterogeneity achieved a
pathological complete response (pCR), whereas 55% of those with homogeneous tumors
did it. Altogether, T-DM1 is hypothesized to fare poorly if tested in the HER2-low setting.
Given this apparent failure of the early anti-HER2 agents for the treatment of HER2-
low BC, why should this niche be revisited with other anti-HER2 agents? Which are the
anti-HER2 agents capable of targeting HER2-low BC cells with clinical efficacy? Are there
combinations capable of overcoming those low levels of HER2 expression?
Cancers 2021, 13, 1015 6 of 18

3. Mechanisms of Action and Clinical Efficacy of the Novel Anti-HER2 Drugs in

HER2-Low BC
The novel agents being tested for the treatment of HER2-low BC patients are catego-
rized as ADCs deploying anti-HER2 epitopes in their monoclonal antibody component,
though with different cytotoxic warheads than trastuzumab–emtansine, and a bi-specific
antibody targeting HER2 and HER3 [46–49]. These novel agents have differential fea-
tures that may explain their in vitro and in vivo activity beyond that seem with earlier
anti-HER2 agents, such as the bystander killing effect of non-target cancer cells with the
novel ADCs, and the bypass of HER3-mediated resistance with zenocutuzumab, the bi-
specific antibody [49,50]. Therefore, their deployment in clinical trials of HER2-low BC is
further explored.

3.1. Trastuzumab-Deruxtecan
Trastuzumab–deruxtecan (T-Dxd) is an ADC, which, apart from sharing the same
HER2 targeting monoclonal antibody, differs from T-DM1 in several aspects (Table 2). Most
importantly, its cleavable linker, with its cell membrane permeable exatecan derivative
(a topoisomerase I inhibitor payload) altogether with a higher drug-antibody ratio (of
8:1), elicits a greater antitumor effect [46]. There are two fronts for that: first, a greater
amount of the cytotoxic payload reaches the targeted cells (i.e., more potent cytotoxic
effect); second, there is the bystander killing effect. This effect can be understood as the
potential (of a given ADC) to kill the antigen-negative, surrounding cells of a targeted
antigen-positive cell, following diffusion of the free cytotoxic moiety from inside the
dead, antigen-positive cells [50]. With that, low/non-expressing HER2 cells in the tumor
vicinity of HER2 overexpressing cells
Cancers are13,also
2021, x affected by T-Dxd, cell-membrane permeable
cytotoxic moiety (Figure 1) [51]. Ultimately, the bystander killing effect has the potential to
Cancers 2021, 13, x Cancersan
induce 2021, 13, x
improved Cancers 2021,
clinical activity 13,setting
in the x 7 of 18
of low or heterogeneous HER2 expression
while maintaining a safe therapeutic index.
Table 2. Comparison of trastuzumab–emtansine (T-DM1) vs. trastuzu
Cancers
Cancers 2021,
2021, 13,
13, xx Cancers 2021, 13, x Cancers 2021, 13, x 77 of
of 18
18
deruxtecan (T-Dxd).
Table
Table Comparison
2. 2. Comparison of of
trastuzumab–emtansine
trastuzumab–emtansine
Table 2. Comparison (T-DM1)
(T-DM1)ofvs. trastuzumab–duocarmazine
Table 2. Comparison(T-DM1)
trastuzumab–emtansine
vs. trastuzumab–duocarmazine (SYD vs.−trastuzumab–duocarmazine
986) vs.
of trastuzumab–emtansine
(SYD−986) vs.trastuzumab–
(T-DM1) vs. (SYD−98
trastuzumab– trastuzu
deruxtecan
deruxtecan (T-Dxd).
(T-Dxd). Cancers 2021, 13, x (T-Dxd).
deruxtecan Antibody-Drug
Cancers Conjugate
2021, 13, x (T-Dxd).
deruxtecan T-DM1
Table
Table 2.2. Comparison
Comparison of of trastuzumab–emtansine
trastuzumab–emtansine
Table 2. Comparison (T-DM1)
(T-DM1) vs. trastuzumab–duocarmazine
HER2 targeting
of trastuzumab–emtansine
vs.Table vehicle
trastuzumab–duocarmazine
2. Comparison (T-DM1) (SYD−986)
of trastuzumab–emtansine
(SYD−986) vs.
vs. trastuzumab–
Trastuzumab
vs. trastuzumab–duocarmazine
trastuzumab–
(T-DM1) vs. (SYD−98
trastuzu
Antibody-Drug
Cancers 2021, 13, x
Antibody-Drug
deruxtecan Conjugate
Conjugate Antibody-Drug T-DM1 Conjugate Antibody-Drug Conjugate
SYD-986
T-DM1
SYD-986 T-DM1
T-Dxd
SYD-986
T-Dxd 7 of 18
deruxtecan (T-Dxd).
(T-Dxd). deruxtecan (T-Dxd). deruxtecan Linker
(T-Dxd). Non-cleavable
HER2targeting
HER2 targetingvehicle
vehicle HER2 targeting
Trastuzumab vehicle HER2 targeting vehicle
Trastuzumab
Drug–antibody Trastuzumab
ratio Trastuzumab
Trastuzumab
Trastuzumab
3.5:1 (T-DM1) vs. (SYD−98
Antibody-Drug Table 2.Trastuzumab
Comparison of trastuzumab–emtansine
Table Trastuzumab
2. Comparison (T-DM1)
of trastuzumab–emtansine Trastuzumab
vs. trastuzumab–duocarmazine trastuzu
Antibody-DrugLinkerConjugate
Conjugate Antibody-Drug T-DM1
T-DM1 Conjugate
Non-cleavable
Linker Antibody-Drug SYD-986
SYD-986
Linker
Cytotoxic
T-DM1
Conjugate
Non-cleavable
Cleavable
moiety
T-Dxd
T-Dxd
T-DM1
SYD-986
Non-cleavable
Cleavable
Maytansine Cleavable
derivative
HER2 Linker
targeting vehicle deruxtecan (T-Dxd).
Non-cleavable
Trastuzumab deruxtecanTrastuzumab
(T-Dxd).
Cleavable Cleavable
Trastuzumab
HER2 targeting vehicle
Drug–antibody HER2 targeting
Trastuzumab
ratio of trastuzumab–emtansine
Drug–antibody 3.5:1vehicle
ratio HER2 targeting
Trastuzumab
Drug–antibody Trastuzumab
vehicle
ratio
2.8:1
3.5:1 Trastuzumab
Trastuzumab
Trastuzumab
3.5:1
8:12.8:1
Table 2. Comparison (T-DM1) vs. Cytotoxic moiety
trastuzumab–duocarmazine MoA Antimicrotubule
(SYD−986) (mitotic poison)
vs. trastuzumab– A
Linker
Drug–antibody
Linker
Cytotoxic ratio
moiety Non-cleavable
Cytotoxic
Maytansine
Antibody-Drug 3.5:1
Non-cleavable
Linker moiety
derivative Antibody-Drug
Conjugate Cleavable
Linker
Cytotoxic
Maytansine 2.8:1
Non-cleavable
Cleavable
moiety
Seco-DUBA
T-DM1 derivative
Conjugate Cleavable
Maytansine
Exatecan 8:1
Non-cleavable
Cleavable
Cleavable
derivative
Seco-DUBA
T-DM1derivative
SYD-986 E
deruxtecan (T-Dxd). Diffusible cytotoxic moiety?
Drug–antibody
Drug–antibody
Cytotoxic
Cytotoxic moiety
moietyratio
ratio
MoA Drug–antibody
Antimicrotubule
Cytotoxic
HER2 3.5:1
3.5:1
moiety
targeting
Maytansine ratio
(mitotic
MoA poison)
vehicle
derivative Drug–antibody
Cytotoxic moiety
Antimicrotubule
HER2 2.8:1
2.8:1
Alkylating
targeting 3.5:1
ratio
MoA
Trastuzumab(mitotic
agent poison)
vehicle
Seco-DUBA Antimicrotubule
Topoisomerase 8:1
3.5:1
8:12.8:1
Alkylating
Trastuzumab(mitotic
I inhibitor
Trastuzumab
Exatecan agent
derivative poison) Topo A
Cytotoxic
Antibody-Drug
Cytotoxic moiety
Conjugate
moiety Maytansine
CytotoxicT-DM1
Maytansine
Linker derivative
moiety
derivative Cytotoxic
Bystander Seco-DUBA
SYD-986
Maytansine
Seco-DUBA
moiety
killing
Linker derivative
effect?
Non-cleavable Exatecan T-Dxd
Maytansine
Exatecan derivative
Seco-DUBA
derivative
derivative
Non-cleavable
Cleavable E
Diffusible
Cytotoxiccytotoxic
moiety MoAmoiety? Diffusible cytotoxic
Antimicrotubule moiety?
(mitotic Diffusible cytotoxic
poison) Alkylating moiety?
agent Topoisomerase I inhibitor
Cytotoxic
HER2 moiety
targeting
Cytotoxic moiety MoA
vehicle
MoA Antimicrotubule
Trastuzumab
Antimicrotubule
Cytotoxic moiety
Drug–antibody (mitotic
MoA poison)
(mitotic
ratio poison) Cytotoxic Alkylating
Trastuzumab
Antimicrotubule
Alkylating
Drug–antibodymoiety agent
MoA
(mitotic
3.5:1agent
ratio poison) Topoisomerase
Trastuzumab
Antimicrotubule
Topoisomerase
Alkylating II inhibitor
(mitotic
3.5:1
2.8:1 inhibitor
agent poison) Topo A
Targets HER2-positive or homog-
Bystander
Diffusible
Diffusible killing
Linker
cytotoxic
cytotoxic effect?
moiety?
moiety? Bystander
Cytotoxickilling
Non-cleavable
moietyeffect? Bystander
Cytotoxic killing
Maytansine
moietyeffect?
Cleavable derivative Cleavable
Maytansine
Seco-DUBA
derivative E
Diffusible cytotoxic moiety? Diffusible cytotoxic moiety? Diffusible enous cytotoxic
tumors? moiety?
TargetsDrug–antibody
HER2-positiveratio or homog-Targets HER2-positive
Cytotoxic 3.5:1 or
moiety MoAhomog-Targets
Targets HER2-positive
Cytotoxic
Antimicrotubule
HER2-low moiety2.8:1MoA
or or homog-
(mitotic
heterogene- poison)
Antimicrotubule 8:1(mitotic
Alkylating agent poison) Topo A
Bystander
Bystander
Bystander killing
killing effect?
effect?
enous killing
Cytotoxic tumors? effect?
moiety Bystander
enous killing
Maytansine
tumors? effect?
derivative
Diffusible cytotoxic moiety? Diffusible
Bystander
enous killing
Seco-DUBA
tumors?
ouscytotoxic
effect?
tumors?moiety?
Exatecan derivative
Targets
Targets HER2-positive
Cytotoxic
HER2-low
Targets moiety
HER2-positive or
or homog-
MoAhomog-
or heterogene-
HER2-positive or Antimicrotubule
Targets HER2-positive
HER2-low or(mitotic poison)
or homog-
heterogene- Alkylating
Targets HER2-positive
HER2-low oragent
homog-
or heterogene- Topoisomerase I inhibitor
Legend: MoA = mechanism of
enous
enous
homogenous
ous tumors?
tumors?
tumors?
tumors? Bystander
enous
ous killing
tumors?
tumors? effect? Bystander
enous
ous killing
tumors?
tumors? effect?
Diffusible cytotoxic moiety?
Targets
Targets HER2-low
HER2-low or
or heterogene-
heterogene- Targets HER2-positive
HER2-low or heterogene- Targets HER2-low or heterogene- Legend:
action.isMoA
Targets
Bystander
HER2-low or
killing effect?
Targets Legend: MoA = Targets
or homog-mechanism of action.
HER2-positive Legend: MoA = mechanism
or homog- In fact,ofT-Dxd not =only
mechanism of
active in
ous
ous tumors?
tumors? ous tumors?
enous tumors? ous tumors?
enous tumors?
heterogeneous tumors? rived xenograft model, but it also retain
Targets HER2-positive or homog-Targets Legend: MoA
not= =mechanism
mechanism of
of action. In
In fact, T-Dxd
HER2-lowLegend:
Legend: oris MoA =only
heterogene-
MoA
active
mechanism
Targets Inof fact,
in
HER2-low a T-Dxd
T-DM1
Legend:isMoA
action. insensitive,
action. or heterogene-
not =only
mechanism
levels offact,
active HER2 T-Dxd
HER2-positive
inof T-DM1
Legend:
action. isMoA
a expression, not =only
patient-de- active
insensitive,
mechanism
unlike HEin
HER
of
T-DM
enous tumors? rived xenograft model, but it rived
ous tumors? also retains
xenograft its activity
ous tumors? butrived
model, against it alsoxenograft
several
retains
BC model,
its
models
activity but
with it also
against
low retain
sever
paralleled in early phase trials, with m
Targets HER2-low or heterogene- levelsIn
Inoffact,
fact,
HER2 T-Dxd
T-Dxd is
is not
not only
expression, only active
active
unlike
levels In
T-DM1in
offact,
in aa T-Dxd
HER2 T-DM1
T-DM1
[46]. insensitive,
isMoA
insensitive,
not
expression,
Legend: Both =only
levels
preclinical
unlikeIn
mechanism
fractory,
HER2-positive
active
HER2-positive
fact,
of in
HER2
T-DM1 T-Dxd
a expression,
observations
of T-DM1
Legend: isMoA
[46].
action.
HER2-positive
patient-de-
patient-de-
notinsensitive,
Both
have=only
BC
active
unlike
preclinical
been
mechanism
patients
HEin
T-DMo
of
co
ous tumors? rived
rived xenograft
xenograft
paralleled in early model,
model,
phase but
but it
trials,also retains
it paralleled
rived
also retains
with inits
xenograft
meaningful activity
itsearly
activity
model,
phaseagainst
against
but
clinicalrived several
itresponses
also
several
paralleled
trials, xenograft
with in BC
retains
BC models
its
earlymodels
model,
meaningful
seen activity
in phase
a with
but
with itlow
against
T-DM1-re-
clinical low
also
trials, sever
retain
with
responsem
levels HER2-low BC patientshave [24,52]. In the lat
levels of
fractory,of HER2
HER2 expression,
Legend:
HER2-positive MoABC
expression, unlike
= mechanism
unlike
levels
patients T-DM1
In
fractory, ofHER2
T-DM1
offact,
cohort [46].
action.
[46].
T-Dxd
HER2-positive
and Both
expression,
Both
is preclinical
preclinical
not
also levels
aunlike
inonly
BC In
fractory, observations
offact,
T-DM1
active
patients
heavily observations
HER2
in a expression,
T-Dxd [46].
T-DM1
HER2-positive
cohort
pretreated is Both
and have
not been
preclinical
unlike
been
insensitive,
only
also
BC
cohort in T-DM
active
patients HE
a heavil
of o
in
co
paralleled metastatic BC patients were enrolled. Th
paralleled in
HER2-low BCearly
in early phase
phase
patients trials,
[24,52]. Inwith
trials,
paralleled
with
rived
HER2-low meaningful
meaningful
in
thexenograft
latter early
BCtrial,
patients
onclinical
phase
model,clinical
but itresponses
trials,
paralleled
responses
rived
its[24,52].
dose also
HER2-low with in
xenograftseen
retains
expansion
In the BC
latter itsin
meaningful
seen
early in
model,
part, aa on
phaseT-DM1-re-
T-DM1-re-
activity
patients
trial,
54 clinical
but trials,
against
[24,52].
HER2-low
its it response
with
also
doseIn m
sever
retain
the lat
expans
In
fractory,fact, T-Dxd
HER2-positiveis notBConly active
patients in a
cohortT-DM1
and dian
insensitive,
also in a duration of
HER2-positive
heavily response
pretreated of
patient-de-
cohort 10.4
of month
fractory, HER2-positive BC enrolled.
patients
fractory,
levels cohort
HER2-positive
ofThe
HER2 and also
expression, BC
inlevels
fractory,
apatients
heavily
unlike HER2-positive
cohort
of T-DM1
HER2pretreatedand
expression,
[46]. cohort
also
Both BC in
patients
of
a heavil
apreclinical
unlike co
metastatic
rived
BC patients were
xenograft
HER2-low BC model,
patients
metastatic
butofit10.4
[24,52]. also
In retains
the
BC
latter
ORRpatients
itstrial,
was
activity
on
37%
were metastatic
against
its dose
(95%
pleenrolled.
CI,
subgroups,
several
expansion
BC
BC
patients
24.3–51.3%),
The ORR
including
models
part, 54
waswere
with 37%
HER2
with
HER2-low
me-
low IHCT-DM
enrolled.
(95% CI, o2
Th
sta
HER2-low
dian durationBC ofpatients [24,52].
response HER2-low
In months
dian the
paralleled latter
durationBC
in trial,
patients
early
(Table
of on its
phase
response
1). [24,52].
T-DXddose
HER2-low
trials,expansion
In
paralleled
dian
of 10.4 the was
with
duration
activity latter
BC
in
months part,
patients
trial,
meaningful
early
of
(Table
seen 54
phase
response on
HER2-low
across[24,52].
1). its
clinicaldose
trials,
of
T-DXd In
10.4
multi- expans
the lat
response
with
monthm
activity
levels of HER2 expression, unlike T-DM1 [46]. Both ing
preclinicaltherapy, and priorwith exposure to a CD
metastatic
metastatic
ple BC
BC patients
subgroups, patients were
includingwere
HER2enrolled.
enrolled.
metastatic
fractory,
pleIHC The
TheBCORR
ORR was
patients
was
HER2-positive
subgroups,
status (2+
including
and 37%
37%
were
1+), BC (95%
metastatic
(95%
fractory,
ple
HER2
HR CI,observations
CI,
enrolled.
patients
subgroups,
status,
IHC 24.3–51.3%),
24.3–51.3%),
BCThepatients
ORR
HER2-positive
cohort
previous
status (2+ and
includingwashave
with
were 37%
also
BC aabeen
HER2
HER2-target-
and 1+), me-
enrolled.
me-
in(95%
patients
HR aIHC CI,Th
co2
heavil
sta
status,
paralleled
dian in
duration early
of phase
response trials,
of 10.4with
monthsmeaningful
(Table 1).clinical
T-DXd two randomized,
responses
activity wasseen
seeninphase
a III
T-DM1-re-
across trials
multi- compa
dian duration of response of 10.4
dian
ing months
duration
HER2-low
therapy, (Table
BC of
and
ing therapy, and prior exposure to a CDK4/6i. Based on choiceresponse
1).
patients
prior T-DXd of
dian
[24,52].
exposure
ing 10.4
activity
HER2-low duration
In months
the
therapy,
to a
these promising was of
(Table
seen
latter
BC response
patients
CDK4/6i.
and across
trial,
prior 1).
on T-DXd
of
multi-
[24,52].
Based
efficacy its
exposure
on 10.4
doseInactivity
thesemonth
expans
the
to
signals, or me a lat
prom
CD
fractory,
ple HER2-positive BC patients cohort and also in in HER2-low,
aenrolled.
heavily pretreated unresectable
cohort ofIHC
ple subgroups,
two subgroups,
randomized, including
including
phase IIIHER2
HER2 pleIHC
IHC
metastatic
trials
two status
subgroups,
comparing BC(2+
status
randomized, (2+ and
including
and
patients
T-DXd
phase1+),
1+),
were HR
HER2
HR
versus
IIIple status,
status,
subgroups,
metastatic
two
trials IHC previous
previous
status
BC
randomized,
chemotherapyThe
comparing patients
ORR HER2-target-
including
(2+ HER2-target-
and
was
phase
T-DXd
of 1+),
were HER2
37%
III
physician’s HR status,
enrolled.
(95%
trials
versus sta
CI,
compa
chemoTh2
HER2-low
ing therapy,
ing therapy,
choice BCandpatients
in HER2-low, prior
and prior [24,52].
exposure
exposure In
ingto
dian
unresectable the
to
choice a latter
CDK4/6i.
therapy,
ain
CDK4/6i.
duration
or trial,
and
metastatic on
Based
Based
prior
of response
HER2-low, its dose
on these
exposure
on ing
these
of
dian expansion
10.4
choice
BCunresectable
are currently promising
therapy,
promising
to a
duration
months
inor part,
CDK4/6i.
and
of
(Table
HER2-low,
ongoing
metastatic 54
efficacy
efficacy
prior HER2-low
Based
response signals,
exposure
1). signals,
on
T-DXd
of
unresectable
[53,54].
BC are these
10.4 to aprom
CD
activity
month
currentlyor meo
on
3.2. Trastuzumab-Duocarmazine
metastatic
two BC patients
two randomized,
randomized, phase
phasewere
III enrolled.
III trials
trials
ple The ORR
comparing
twosubgroups,
comparing
randomized, was
T-DXd
T-DXd
phase
including 37%
versus
versus
III (95%
two
trials
HER2
ple CI, 24.3–51.3%),
chemotherapy
chemotherapy
randomized,
comparing
subgroups,
IHC of
phase
T-DXd
ofand
statusincluding
(2+ with atrials
physician’s
physician’s
IIIversus
1+),
HER2 me-
HRIHC compa
chemo
status,
sta
dian duration
choice in of
HER2-low, response
choice in HER2-low, unresectableof 10.4
unresectable months
choiceor (Table
metastatic
or in
metastatic
HER2-low, 1).
BC T-DXd
are currently
BCunresectable
are currently Trastuzumab–duocarmazine
activity
choice inor was
ongoing
ongoing seen
HER2-low,
metastatic across
[53,54].
[53,54]. multi-
unresectable
BC are currently (SYD
or meo
 potential (of a given ADC) to kill the antigen-negative, surrounding cells of a targeted
antigen-positive cell, following diffusion of the free cytotoxic moiety from inside the dead,
antigen-positive cells [50]. With that, low/non-expressing HER2 cells in the tumor vicinity
of HER2 overexpressing cells are also affected by T-Dxd, cell-membrane permeable cyto-
Cancers 2021, 13, 1015 toxic moiety (Figure 1) [51]. Ultimately, the bystander killing effect has the potential
7 of 18to
induce an improved clinical activity in the setting of low or heterogeneous HER2 expres-
sion while maintaining a safe therapeutic index.

Figure
Figure Schematic
1. 1. Schematic representation
representation ofofHER2-low
HER2-low breast
breastcancer
cancerbeing
being exposed
exposed toto
trastuzumab–emtansine
trastuzumab–emtansine (T-DM1;
(T-DM1; toptopofof
figure), with
figure), itsits
with non-cleavable
non-cleavablelinker
linker attaching
attachingtrastuzumab
trastuzumab toto
the cytotoxic
the maytansine
cytotoxic maytansinederivative,
derivative,andandtototrastuzumab–
trastuzumab–
deruxtecan(T-Dxd;
deruxtecan (T-Dxd;thethebottom
bottomof of figure),
figure), with its cleavable
cleavablelinker
linkerattaching
attachingtrastuzumab
trastuzumabtoto thethe
diffusible,
diffusible,cytotoxic exa-
cytotoxic
tecan derivative. While DM1 is trapped inside the trastuzumab-targeted cells, Dxd is freely diffusible
exatecan derivative. While DM1 is trapped inside the trastuzumab-targeted cells, Dxd is freely diffusible and able to kill and able to kill non-
expressing HER2-cells.
non-expressing Ultimately,
HER2-cells. Ultimately, the the
bystander killing
bystander effect
killing represented
effect herehere
represented explains the success
explains of T-Dxd
the success (and (and
of T-Dxd also of
also of trastuzumab–duocarmazine) in targeting HER2-low tumours, despite their lower degree and heterogeneity ofex-
trastuzumab–duocarmazine) in targeting HER2-low tumours, despite their lower degree and heterogeneity of HER2
pression.
HER2 expression.

In fact, T-Dxd is not only active in a T-DM1 insensitive, HER2-positive patient-derived

xenograft model, but it also retains its activity against several BC models with low levels of
HER2 expression, unlike T-DM1 [46]. Both preclinical observations have been paralleled in
early phase trials, with meaningful clinical responses seen in a T-DM1-refractory, HER2-
positive BC patients cohort and also in a heavily pretreated cohort of HER2-low BC
patients [24,52]. In the latter trial, on its dose expansion part, 54 HER2-low metastatic BC
patients were enrolled. The ORR was 37% (95% CI, 24.3–51.3%), with a median duration
of response of 10.4 months (Table 1). T-DXd activity was seen across multiple subgroups,
including HER2 IHC status (2+ and 1+), HR status, previous HER2-targeting therapy, and
prior exposure to a CDK4/6i. Based on these promising efficacy signals, two randomized,
phase III trials comparing T-DXd versus chemotherapy of physician’s choice in HER2-low,
unresectable or metastatic BC are currently ongoing [53,54].

3.2. Trastuzumab-Duocarmazine
Trastuzumab–duocarmazine (SYD985) is a novel ADC in earlier phases of clinical
development, composed of trastuzumab, a cleavable linker, and a DNA-alkylating duo-
carmycin warhead [47]. Its drug–antibody ratio is 2.8:1, and the cytotoxic moiety is actually
a cell-permeable pro-drug (seco-duocarmycin-hydroxybenzamide-azaindole (seco-DUBA)),
cleaved into the active toxin (DUBA) in intracellular lysosomes by proteases, following
Cancers 2021, 13, 1015 8 of 18

internalization. Interstitial cleavage of trastuzumab–duocarmazine by malignant cells

secreting cathepsin B also occurs, generating free DUBA capable of inducing the desired by-
stander killing effect (Table 2) [50,55]. Despite its lower drug–antibody ratio, trastuzumab–
duocarmazine has been shown significantly more potent than T-DM1 in comparative essays
of HER2-low, patient-derived xenograft BC models [47].
Early clinical signs of efficacy with trastuzumab–duocarmazine have already been
demonstrated in all levels of HER2 IHC expression in a first-in-human trial, albeit thus far
the only phase 3 trial recruiting is restricted to HER2-positive MBC patients (clinicaltrial.gov
identifier: NCT03262935) [25]. On its first-in-human study, trastuzumab–duocarmazine
was tested on treatment-refractory, locally advanced or metastatic tumors in order to
assess safety, pharmacokinetics and preliminary tumor activity. Forty-seven HER2-low BC
patients were enrolled in the BC dose expansion cohort. Six of 15 HR-negative (40%, 95%
CI 16.3–67.6) and 9 of 32 (28%, 95% CI 13.8–46.8) HR-positive patients achieved a partial
response (Table 1).

3.3. XMT-1522
XMT-1522 inaugurated a generation of HER2-targeted ADCs with distinct structural
features: instead of trastuzumab, XMT-1522 uses HT-19, which binds a different HER2
epitope than trastuzumab, linked via a biodegradable cysteine-linkage to an auristatin-
derivative, yet another anti-tubulin agent. XMT-1522 has the highest drug–antibody ratio
of the field (12:1), elicits a controlled bystander killing effect, and has been shown to
outperform T-DM1 in HER2-positive and low xenograft cancer models [48]. Despite
preliminary signs of clinical efficacy, XMT-1522 development was halted at phase I due to
commercial reasons [56].

3.4. Zenocutuzumab
Not only ADCs are being tested in HER2-low metastatic BC patients. Currently, the
bi-specific humanized IgG1 antibody zenocutuzumab (MCLA128) is undergoing clinical de-
velopment, with results from phase II trials available [26,57]. With the novel dock and block
effect, zenocutuzumab works by docking to HER2 domain I, positioning the anti-HER3 arm
fit to block the domain III of HER3, such as to prevent the binding of any activating ligand
(e.g., heuregulin) [49]. HER2/HER3 heterodimerization is, therefore, inhibited altogether
with the subsequent oncogenic intracellular signaling cascade. Zenocutuzumab is more
potent than pertuzumab in inhibiting HER2/HER3 heterodimerization, including at higher
heuregulin concentrations, and is also capable of eliciting ADCC [49].
In HR-positive/HER2-low BC, bidirectional crosstalk between the estrogen receptor
(ER) and the HER2/HER3 axis can drive ET resistance, whereby upregulation of heureg-
ulin and HER2/HER3 heterodimers can phosphorylate the ER, and ER signaling can
upregulate HER2 and HER3 expression [18,39]. In this sense, zenocutuzumab with ET
has been demonstrated to sustain a better antitumor effect than ET alone in HER2-low BC
xenograft models [49]. In a phase I/II trial enrolling HER2-positive BC and other epithelial
tumors, the zenocutuzumab recommended phase II dose was determined and shown to
be remarkably safe, with very few grade 3–4 adverse events (AEs). Afterwards, this dose
was tested in a phase 2, single-arm trial of endocrine-resistant, ER-positive/HER2-low BC
patients, who had experienced disease progression while on a cyclin-dependent kinase
4 and 6 inhibitor (CDK4/6i) [26,57]. Fifty patients were treated with zenocutuzumab
plus ET (fulvestrant or an aromatase inhibitor), and 8 sustained a clinical benefit at week
24 (clinical benefit rate of 16.7% (90% CI, 8.6–28.1)), with one patient showing a partial
response followed by a long-lasting disease stabilization (Table 1).

4. Exploiting Combination Treatments in HER2-Low Breast Cancer

Combination strategies in HER2-low BC with anti-HER2 agents that are currently
being tested include immune checkpoint inhibitors (ICIs), CDK4/6i, ET, among others. This
Cancers 2021, 13, 1015 9 of 18

effort includes both the deployment of novel anti-HER2 agents as well as the repurposing
of older drugs.

4.1. Immunotherapy
Preclinical and clinical data suggest that HER2-positive tumors are immunogenic [58].
HER2-positive tumors have a higher mutational burden (2.05 mutations/Mb) compared
to luminal tumors (up to 1.38 mutations/Mb) [59]. They also contain a higher number of
tumor-infiltrating lymphocytes (TILs) and higher PD-L1 expression [58,59]. The lympho-
cytic infiltrate observed in primary HER2-positive tumors has been associated with pCR
and improved survival outcomes [60]. Moreover, ADCC is a key player when it comes to
modulating the effect of trastuzumab [58]. Preclinical studies suggest that the combination
of trastuzumab with drugs targeting immune checkpoints could overcome trastuzumab
resistance [60]. High expression of programmed cell death protein 1 (PD-1), PD-L1 and
other checkpoint molecules has been observed in this setting [58].
Several chemotherapeutic agents, by inducing tumor cell death and immunogenic cell
death, also activate the immune system [61]. Topoisomerase inhibitors have successfully
been combined with ICIs in syngeneic mouse models, but there is a concern that they
could induce lymphopenia and thus attenuate the effect of ICIs [61]. In this sense, T-Dxd
could be an ideal partner for ICI by combining trastuzumab-induced ADCC with the
topoisomerase I inhibitor warhead-induced immunogenic cell-death and the improved
therapeutic index [62]. In preclinical studies, T-Dxd increased tumor-infiltrating dendritic
cells, upregulated their CD86 expression in vivo, increased tumor-infiltrating CD8+ T cells
and enhanced PD-L1 and major histocompatibility complex class I expression on tumor
cells [62]. Furthermore, in mouse models, combination therapy with T-Dxd and an anti-PD-
1 antibody was more effective than monotherapy, possibly due to increased T-cell activity
and upregulated PD-L1 expression induced by T-Dxd [62].
In a phase 1b study, 16 patients with treatment-refractory, HER2-low BC were treated
with the combination of T-Dxd and the anti-PD-1 nivolumab [38]. Overall, treatment with
T-Dxd + nivolumab had a manageable safety profile and showed preliminary encouraging
efficacy data, albeit similar to that of T-Dxd monotherapy (Table 1).
Altogether, despite preliminary, these data support the rationale for combining anti-
HER2 therapies with ICI in trials of HER2-low BC patients (Table 3).

Table 3. Ongoing combinations trials with immune checkpoint inhibitors and the novel anti-HER2 agents in HER2-low
breast cancer.

ClinicalTrials.gov
Drugs Tested Study Design Patient Population Primary Endpoint Status
Identifier
Phase Ib, open-label, Advanced BC
Trastuzumab– two-part, (HER2-positive and
Phase I: MTD
deruxtecan + multicenter, HER2-low) and Recruiting NCT04042701
Phase II: ORR
pembrolizumab nonrandomized, HER2-positive
multiple-dose NSCLC
Advanced BC
Trastuzumab–
Phase Ib, multicenter, (HER2-positive and Phase I: MTD
deruxtecan + Ongoing NCT03523572
two-part, open-label HER2-low) and Phase II: ORR
nivolumab
urothelial cancer
Phase Ib/II,
Trastuzumab– Arm 6: Advanced
two-stage,
deruxtecan + TNBC with low Safety Recruiting NCT03742102
open-label,
durvalumab HER2 expression
multicenter
Trastuzumab– Phase Ib, open-label,
deruxtecan + modular, Module 2: advanced Safety and
Not yet recruiting NCT04556773
durvalumab + dose-finding and HER2-low BC tolerability
paclitaxel dose-expansion
Legend: BC: breast cancer; ET: endocrine therapy; NSCLC: non-small cell lung cancer; MTD: maximum tolerated dose; ORR: objective
response rate; TNBC: triple-negative breast cancer.
Cancers 2021, 13, 1015 10 of 18

4.2. Endocrine Therapies

Building upon the hypothesis of tumor resistance development via bidirectional
crosstalk between HER2/HER3 and ER axes, Collins et al. used a xenograft mouse model
of ER-positive/HER2-low human BC to evaluate a triple therapy targeting HER2, HER3,
and ER [63]. It was found that the addition of fulvestrant significantly enhanced antitumor
responses to the HER2 and 3 targeting agents. In this sense, as mentioned previously, the
addition of the HER2/HER3 bi-specific antibody zenocutuzumab to ET showed clinical
activity in ET and CDK4/6i-refractory patients, resulting in an endocrine-sensitivity rescue
of 17% of them (Table 1) [26].
Co-targeting of the ER and HER2 axis is also being attempted in a phase Ib trial with
T-Dxd and anastrozole or fulvestrant [64].

4.3. CDK4/6i
As a downstream pathway to HER2, deregulation of the cyclin D1-CDK4 axis is a
common mediator of HER2 therapy resistance [65]. Moreover, preclinical evidence suggests
that CDK4/6i may hold activity beyond luminal BC: not only CDK4/6i are effective in
lapatinib-resistant, HER2-positive cell models, but their use altogether with anti-HER2
agents has a synergistic effect [66,67]. CDK4/6i sensitizes lapatinib-resistant cell lines to
HER2-targeted therapies, leading to better inhibition of cell proliferation and, in patient-
derived xenograft tumors and transgenic mice models, to improved control of tumor
growth and delays tumor recurrences, respectively.
In order to test a chemotherapy-free approach in the early disease setting, the NA-
PHER2 trial, an open-label, exploratory, phase II study, evaluated neoadjuvant pertuzumab,
trastuzumab, fulvestrant and the CDK4/6i palbociclib [37]. It included 23 patients with
HR-positive/HER2-low BC with a Ki67 > 20% in one of its cohorts (cohort C), with 2
co-primary endpoints: on-treatment changes of baseline Ki67 to week 2 and at surgery
(16 weeks). A robust Ki67 decrease was demonstrated, especially at week 2, as well as 18
(78.3%) objective responses were seen, underlining the clinical potential of the combination.
However, unlike cohort A and B, where only HER2-positive BC cases were enrolled, no
patient achieved a pCR (Table 1) [68].

4.4. Other Combinations

A few other treatment combinations are being tested with T-Dxd in phase 1, mul-
ticohort clinical trial, including chemotherapy agents and AKT inhibitors, based on the
premise of distinct and non-overlapping, cytotoxic mechanisms resulting in additional
efficacy [64]. Hitherto, phase 1 data are still being generated in order to evaluate the safety
of these distinct combinations (clinicaltrial.gov identifier: NCT04556773).

5. Safety
The safety profile of the new different compounds tested for HER2-low BC is inter-
estingly heterogeneous, according to the cytotoxic warhead, in the case of ADCs, or to
the immunogenicity capacity, in the case of bispecific antibodies (Figure 2). Thus, upon
regulatory approval of these novel agents, clinicians will be able to choose according to
patient comorbidities and preferences in case of comparable efficacy.
Cancers 13, 1015
2021,2021,
Cancers 13, x 11 of 18 11 of 18

Figure 2. Incidence
Figure of key
2. Incidence adverse
of key adverseevents
eventsinduced
induced by by trastuzumab–deruxtecan, trastuzumab–duocarmazine
trastuzumab–deruxtecan, trastuzumab–duocarmazine and zeno-
and ze-
cutuzumab. Abbreviations: T-DXd: trastuzumab–deruxtecan, SYD-985: trastuzumab–duocarmazine, MCLA-128: zeno-
nocutuzumab. Abbreviations: T-DXd: trastuzumab–deruxtecan, SYD-985: trastuzumab–duocarmazine, MCLA-128: ze-
nocutuzumab,
cutuzumab, LVEF:LVEF: left ventricular
left ventricular ejectionfraction.
ejection fraction. §§ Data
Dataextracted from
extracted all enrolled
from patients
all enrolled (dose-escalation
patients and
(dose-escalation and
dose-expansion parts). * The only reported ocular adverse event was dry eye (data extracted from all enrolled patients
dose-expansion parts). * The only reported ocular adverse event was dry eye (data extracted from all enrolled patients
(dose-escalation and dose-expansion parts)). ** other non-reported ocular adverse events include episcleritis (3%), corneal
(dose-escalation andretinal
toxicity (1%) and dose-expansion
hemorrhageparts)).
(1%). ** other non-reported ocular adverse events include episcleritis (3%), corneal
toxicity (1%) and retinal hemorrhage (1%).
5.1. Hematological Toxicity
Despite their targeted cytotoxicity, all ADCs share some hematological AEs [69]. In
5.1. Hematological Toxicity
the phase 2 study on T-Dxd, the following all-grade hematological AEs were reported:
Despite (35%),
neutropenia their targeted cytotoxicity,
anemia (30%) all ADCs share
and thrombocytopenia some
(21%) hematological
[52]. AEs [69]. In
Grade 3 or higher
the phase 2 study on T-Dxd, the following all-grade hematological AEs
hematological AEs were observed for neutropenia (20%), anemia (8%), leukopenia (6%) were reported:
and thrombocytopenia (4%). Febrile neutropenia occurred in 2% of patients.
neutropenia (35%), anemia (30%) and thrombocytopenia (21%) [52]. Grade 3 or higher In the dose-
expansion part AEs
hematological of the phase
were 1 study of
observed fortrastuzumab–duocarmazine,
neutropenia (20%), anemia there were
(8%), neutro-
leukopenia (6%)
penia (10% grade 1–2 and 6% grade 3), anemia (9% grade 1–2 and 1% grade 3),
and thrombocytopenia (4%). Febrile neutropenia occurred in 2% of patients. In the dose- thrombo-
cytopenia part
expansion (5% grade
of the1–2 and11%
phase grade
study of 3), lymphopenia (5% grade 1–2 there
trastuzumab–duocarmazine, and 1% weregrade 3)
neutropenia
and pancytopenia AEs (1% grade 3) [25].
(10% grade 1–2 and 6% grade 3), anemia (9% grade 1–2 and 1% grade 3), thrombocytope-
nia (5% grade 1–2 and 1% grade 3), lymphopenia (5% grade 1–2 and 1% grade 3) and
5.2. Hepatic Toxicity
pancytopenia AEs (1% grade 3) [25].
All ADCs, including those already used in clinical practice, such as T-DM1, are po-
tentially
5.2. Hepatichepatotoxic
Toxicity [70,71]. In the phase 2 study of T-DXd, AST and ALT increase was
observed in 15% and 12% of patients (all-grade), respectively; grade 3 elevations occurred
All
in 2% of ADCs, including
cases [52]. In the phasethose already used
I dose-expansion inofclinical
part practice, such as T-DM1,
trastuzumab–duocarmazine, he- are
potentially hepatotoxic [70,71]. In the phase 2 study of T-DXd, AST and
patic AEs were AST increase (5% grade 1–2 and 1% grade 3), GGT increase (3% grade 1– ALT increase was
observed
2, 1% gradein 4)
15%andand 12% phosphatase
alkaline of patients (all-grade),
increase (2%respectively;
grade 1–2 andgrade 3 elevations
1% grade occurred
3) [25]. One
inpatient
2% ofdied
cases [52].
from In the
hepatic phasealthough
failure, I dose-expansion
this death waspart
notofconsidered
trastuzumab–duocarmazine,
treatment-re-
lated. AEs were AST increase (5% grade 1–2 and 1% grade 3), GGT increase (3% grade 1–2,
hepatic
1% grade 4) and alkaline phosphatase increase (2% grade 1–2 and 1% grade 3) [25]. One
5.3. Gastrointestinal
patient Toxicityfailure, although this death was not considered treatment-related.
died from hepatic

5.3. Gastrointestinal Toxicity

Due to its topoisomerase inhibitor warhead, T-Dxd is characterized by a comparatively
worse gastrointestinal (GI) toxicity profile [72]. Gastrointestinal AEs were reported among
the most common T-Dxd-induced AEs and included nausea (78% all grades, 8% grade 3),
vomiting (46% all grades, 4% grade 3), constipation (36% all grades), decreased appetite
Cancers 2021, 13, 1015 12 of 18

(31% all grades, 2% grade 3), diarrhea (29% all grades, 3% grade 3) and abdominal pain (17%
all grades, 1% grade 3) [52]. Regarding trastuzumab–duocarmazine, the most common GI
AEs were decreased appetite and nausea, reported in less than 20% of patients; the only
grade 3 gastrointestinal AEs reported were diarrhea and decreased appetite (both 1% in
the dose-expansion cohort) [25].

5.4. Pulmonary Toxicity

Albeit uncommon, interstitial lung disease (ILD) is potentially life-threatening, and
patients treated with HER2-targeting ADCs should be carefully monitored, according to
the previous experience with T-DM1 [70,71]. Likewise, in a phase 1b trial, three toxic deaths
occurred with T-DXd: one case of ILD and two cases of pneumonitis. Globally, eleven
ILD were centrally re-evaluated, and eight of them were considered T-DXd-induced. The
most common AEs leading to treatment discontinuation were pneumonitis (n = 7) and
ILD (n = 3). Grade 2 or higher ILD were treated with steroids [24]. In the phase 2 study of
T-DXd in HER2-positive BC, ILD was reported for 14% of patients [52]. In a pooled analysis
of ILD data coming from seven trials of T-DXd, among 665 patients, 66 (9.9%) developed
ILD, with 13 (2%) of grade ≥3, 5 (<1%) deaths, and with a median (range) time to onset of
149 days (16–596 days) [73]. Most grade ≥2 cases were managed with steroids or steroids
plus antibiotics. A higher dose of T-Dxd (6.4 mg/kg, instead of the recommended phase II
dose of 5.4 mg/kg) and Japanese origin seem to be risk factors for ILD.
In the dose-escalation phase for trastuzumab–duocarmazine, one fatal pneumonitis
was reported as a dose-limiting toxicity (2.4 mg/kg), but the risk of pneumonitis diminished
at the recommended phase 2 dose of 1.2 mg/kg q3w [25]. Further analyses are required to
better understand the underlying mechanism and risk factors for the novel ADC-induced
ILD, as well as how to properly manage it.

5.5. Ocular Toxicity

Ocular toxicities dominate the toxicity profile of trastuzumab–duocarmazine [25].
Conjunctivitis was reported in up to 30% of patients in the dose-expansion part of the
phase 1 trial. Other reported ocular AEs were dry eyes, keratitis and blurred vision. Four
patients (3%) had grade 3 conjunctivitis in the dose-expansion cohort. Dose-reduction,
decrease in the frequency of administration, or prophylactic use of eye drops did not
seem to impact these AEs. Nonetheless, most patients were able to continue trastuzumab–
duocarmazine and most ocular problems were reported to improve, therefore, suggesting
that these AEs are manageable.

5.6. Cardiotoxicity
Cardiotoxicity represents a potential risk for patients treated with new HER2-targeting
agents, based on the previous experience gathered from older anti-HER2 agents [74]. Briefly,
HER2-rich cardiomyocytes rely on HER2 growth signaling to maintain their homeostasis
and endure oxidative stress, such as that induced by anthracyclines, for which most BC
patients are exposed through the course of their disease. Upon blockade of this important
survival pathway, cardiotoxicity may follow, which includes mainly decreases in left
ventricular ejection fraction (LVEF). QT interval prolongation has also been reported. In
the phase II study of T-DXd on HER2-positive metastatic BC, LVEF decrease incidence was
low (1.6%): two patients had grade 2, and one had a grade 3 event. All patients recovered
after treatment interruption. QT interval prolongation of any grade was reported for 9
patients (4.9%) and of grade 3 in 2 patients (1.1%) [52]. In the dose-expansion phase I study
of trastuzumab-duocarmazine, an LVEF decrease was observed in 10 patients (7%) with
grade 1–2 and in 1 patient (3%) with grade 3. In 8 patients (5%), an absolute decrease of at
least 10% from baseline to a value below 50% was reported [25].
With zenocutuzumab, no treatment-related cardiotoxicities of clinical relevance were
reported in both early phase trials [26,57]. Although the number of treated patients is
smaller comparing to the trastuzumab-containing ADCs, this encouraging cardiac safety
Cancers 2021, 13, 1015 13 of 18

data likely reflects the improved in vitro cardiomyocyte viability with zenocutuzumab, as
compared to trastuzumab [49].

5.7. Neuropathy
Unlike with the anti-tubulin containing T-DM1, neuropathy seems rare with the novel
ADCs [70,71]. This is due to the different mechanism of action of their cytotoxic moieties,
which does not induce functional disruption of the microtubules in the peripheral neurons.

5.8. Infusion-Related Reactions

Infusion-related reactions (IRR) spam mild reactions, like pyrexia, rash, and flushing,
to severe reactions, i.e., overt anaphylactic shock [75]. They seem to be more frequent with
zenocutuzumab than with the novel ADCs, for which the overall incidence of IRR is less
than 10% [26,57].
In the early phase trials of zenocutuzumab, all-grade IRR incidence ranged from 18%
to 36%, with only a few grade 3–4 IRR (4%). Nonetheless, this has prompted the mandatory
use of prophylactic anti-histaminic, anti-pyretic, and corticosteroid prior to zenocutuzumab
infusion once the recommended phase 2 dose was determined.

6. Conclusions
Three decades after the characterization of the oncogenic HER2 protein, anti-HER2
therapies have just started to advance towards the field of HER2-low BC treatment. Inter-
national guidelines currently recommend a binary model (HER2-positive vs. negative) to
guide clinicians in treatment decisions. However, a great proportion of patients (≈40–50%)
classified as HER2-negative are, in fact, HER2-low, a population at a high unmet medical
need. Despite past drawbacks with older drugs, a new generation of anti-HER2 agents has
recently shown encouraging signs of clinical activity and safety in HER2-low disease.
Due to the retention of all trastuzumab antitumor properties, associated with an
improved tumor-specific cytotoxic effect, as well as the bystander killing effect, ADCs like
T-Dxd and trastuzumab–duocarmazine are able to target and kill HER2-expressing BC cells
even upon low-level of HER2 expression, a once limiting step for the clinical activity of
anti-HER2 agents.
Despite some undisputed successes and promising expectations coming from the
new anti-HER2 agents in HER2-low BC, to the point where two phase 3 trials are already
ongoing, this new treatment strategy underlines the steep road of cancer drug development,
characterized by complex technologies, the important commitment of multiple stakeholders
and, oftentimes, clinical outcomes not always fulfilling preclinical expectations.
Albeit having an improved therapeutic index than traditional chemotherapies, anti-
HER2 ADCs still retain some class-related AEs, many in common with the general profile
of chemotherapeutic agents (myelotoxicity, hepatotoxicity) and trastuzumab profile (car-
diotoxicity), but some depending on the class of the cytotoxic warhead (mainly GI toxicity
with T-Dxd and ocular toxicity with trastuzumab–duocarmazine), altogether with poten-
tially life-threatening AEs, such as ILD. A better understanding of the pathophysiology
of such AEs, altogether with the delineation of risk factors, prevention, and treatment
measures, will further improve the safety profile of these ADCs.
Overall, T-Dxd may be the first HER2-targeted therapy approved for HER2-low BC
patients, based on the strong preclinical rationale, encouraging early efficacy signs hitherto
discussed, and its current stage of development. To overcome the current standard of care,
other than leveraging what has been observed in early phase trials, the two-phase 3 clinical
trials currently ongoing must be able to decrease the occurrence and severity of ILD thus
far observed with T-Dxd, with replicability in the real world.
A further step in the development of HER2-low BC treatment is coming from the
evaluation of new treatment combination strategies. Considering ADC’s ability to induce
immunogenic cell death and thereby an immune-hot tumor microenvironment, further
results from studies evaluating ADCs in combination with ICIs are eagerly expected.
Cancers 2021, 13, 1015 14 of 18

Combined with ET, new anti-HER2 agents, like zenocutuzumab, could provide a new,
chemotherapy-free approach for patients with endocrine-resistant HER2-low BC.
In summary, where trastuzumab and older anti-HER2 agents have left a niche, the
new anti-HER2 agents may succeed. Collectively, these early trial results are building the
foundations for the exciting new field of HER2-low BC treatment.

Author Contributions: D.E.: conceptualization, methodology, investigation, writing—original draft,

writing—review and editing, visualization. E.A.: investigation, writing—original draft. R.S.-C.:
investigation, writing—original draft. E.d.A.: writing—review and editing, supervision. All authors
have read and agreed to the published version of the manuscript.
Funding: This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Conflicts of Interest: The authors have no known competing financial interests or personal rela-
tionships that could have appeared to influence the work reported in the manuscript. Daniel Eiger
reports having received speaker honoraria from Janssen, personal research funding for his ESMO
Fellowship from Novartis, his salary is currently paid by Roche, and he has Roche stocks. Elisa
Agostinetto reports no conflict of interest. Rita Conde reports no conflict of interest. Evandro de
Azambuja reports having received honoraria and the advisory board for Roche/GNE, Novartis,
Seattle Genetics, Zodiac, and Libbs. He reports having received travel grants from Roche/GNE, GSK,
and Novartis. He reports having received institutional grants for his institute from Roche/GNE,
Astra-Zeneca, Novartis, and Servier.

Abbreviations
ADC: antibody-drug conjugate; BC: breast cancer; CBR: clinical benefit rate; CDK 4/6i: cyclin-
dependent kinase 4/6 inhibitors; CI: confidence interval; CR: complete response; DoR: duration of
response; ER: estrogen receptor; ET: endocrine therapy; GM-CSF: granulocyte macrophage-colony
stimulating factor; HR: hazard ratio; iDFS: invasive disease-free survival; IHC: immunohistochem-
istry; LVEF: left ventricular ejection fraction; MCLA-128: zenocutuzumab; MoA: mechanism of
action; MTD: maximum tolerated dose; NE: non-evaluable; NSCLC: non-small cell lung cancer; ORR:
overall response rate; PFS: progression-free survival; PR: partial response; RFI: relapse-free interval;
SD: stable disease; SYD−986: trastuzumab–duocarmazine; T-DM1: trastuzumab–emtansine; T-Dxd:
trastuzumab–deruxtecan; TNBC: triple-negative breast cancer.

References
1. Mendes, D.; Alves, C.; Afonso, N.; Cardoso, F.; Passos-Coelho, J.L.; Costa, L.; Andrade, S.; Batel-Marques, F. The benefit of
HER2-targeted therapies on overall survival of patients with metastatic HER2-positive breast cancer–a systematic review. Breast
Cancer Res. 2015, 17, 140. [CrossRef]
2. Viani, G.A.; Afonso, S.L.; Stefano, E.J.; De Fendi, L.I.; Soares, F.V. Adjuvant trastuzumab in the treatment of her-2-positive early
breast cancer: A meta-analysis of published randomized trials. BMC Cancer 2007, 7, 153. [CrossRef]
3. Swain, S.M.; Miles, D.; Kim, S.-B.; Im, Y.-H.; Im, S.-A.; Semiglazov, V.; Ciruelos, E.; Schneeweiss, A.; Loi, S.; Monturus, E.; et al.
Pertuzumab, trastuzumab, and docetaxel for HER2-positive metastatic breast cancer (CLEOPATRA): End-of-study results from a
double-blind, randomised, placebo-controlled, phase 3 study. Lancet Oncol. 2020, 21, 519–530. [CrossRef]
4. Slamon, D.J.; Clark, G.M.; Wong, S.G.; Levin, W.J.; Ullrich, A.; McGuire, W.L. Human breast cancer: Correlation of relapse and
survival with amplification of the HER-2/neu oncogene. Science 1987, 235, 177–182. [CrossRef]
5. Ross, J.S.; Fletcher, J.A. The HER-2&sol;neuOncogene in Breast Cancer: Prognostic Factor, Predictive Factor, and Target for
Therapy. Stem Cells 1998, 16, 413–428. [CrossRef]
6. Mass, R.; Press, M.; Anderson, S.; Murphy, M.; Slamon, D. Improved survival benefit from Herceptin (trastuzumab) in patients
selected by fluorescence in situ hybridization (FISH). Proc. Am. Soc. Clin. Oncol. 2001, 20, 22a-abstract.
Cancers 2021, 13, 1015 15 of 18

7. Vogel, C.L.; Cobleigh, M.A.; Tripathy, D.; Gutheil, J.C.; Harris, L.N.; Fehrenbacher, L.; Slamon, D.J.; Murphy, M.; Novotny, W.F.;
Burchmore, M.; et al. Efficacy and Safety of Trastuzumab as a Single Agent in First-Line Treatment of HER2-Overexpressing
Metastatic Breast Cancer. J. Clin. Oncol. 2002, 20, 719–726. [CrossRef] [PubMed]
8. Mass, R.D.; Press, M.F.; Anderson, S.; Cobleigh, M.A.; Vogel, C.L.; Dybdal, N.; Leiberman, G.; Slamon, D.J. Evaluation of Clinical
Outcomes According to HER2 Detection by Fluorescence In Situ Hybridization in Women with Metastatic Breast Cancer Treated
with Trastuzumab. Clin. Breast Cancer 2005, 6, 240–246. [CrossRef] [PubMed]
9. Wolff, A.C.; Hammond, M.E.H.; Allison, K.H.; Harvey, B.E.; Mangu, P.B.; Bartlett, J.M.; Bilous, M.; Ellis, I.O.; Fitzgibbons, P.;
Hanna, W.; et al. Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer. Arch. Pathol. Lab. Med. 2018, 142,
1364–1382. [CrossRef]
10. Denduluri, N.; Chavez-MacGregor, M.; Telli, M.L.; Eisen, A.; Graff, S.L.; Hassett, M.J.; Holloway, J.N.; Hurria, A.; King, T.A.;
Lyman, G.H.; et al. Selection of Optimal Adjuvant Chemotherapy and Targeted Therapy for Early Breast Cancer: ASCO Clinical
Practice Guideline Focused Update. J. Clin. Oncol. 2018, 36, 2433–2443. [CrossRef]
11. Cardoso, F.; Senkus, E.; Costa, A.; Papadopoulos, E.; Aapro, M.; André, F.; Harbeck, N.; Lopez, B.A.; Barrios, C.; Bergh, J.; et al.
4th ESO–ESMO International Consensus Guidelines for Advanced Breast Cancer (ABC 4). Ann. Oncol. 2018, 29, 1634–1657.
[CrossRef]
12. Cardoso, F.; Kyriakides, S.; Ohno, S.; Penault-Llorca, F.; Poortmans, P.; Rubio, I.; Zackrisson, S.; Senkus, E. Early breast cancer:
ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 2019, 30, 1194–1220. [CrossRef] [PubMed]
13. Schalper, K.A.; Kumar, S.; Hui, P.; Rimm, D.L.; Gershkovich, P. A Retrospective Population-Based Comparison of HER2
Immunohistochemistry and Fluorescence In Situ Hybridization in Breast Carcinomas: Impact of 2007 American Society of Clinical
Oncology/ College of American Pathologists Criteria. Arch. Pathol. Lab. Med. 2014, 138, 213–219. [CrossRef]
14. Lal, P.; Salazar, P.A.; Hudis, C.A.; Ladanyi, M.; Chen, B. HER-2 Testing in Breast Cancer Using Immunohistochemical Analysis
and Fluorescence In Situ Hybridization: A Single-Institution Experience of 2,279 Cases and Comparison of Dual-Color and
Single-Color Scoring. Am. J. Clin. Pathol. 2004, 121, 631–636. [CrossRef]
15. Giuliani, S.; Ciniselli, C.M.; Leonardi, E.; Polla, E.; DeCarli, N.; Luchini, C.; Cantaloni, C.; Gasperetti, F.; Cazzolli, D.; Berlanda,
G.; et al. In a cohort of breast cancer screened patients the proportion of HER2 positive cases is lower than that earlier reported
and pathological characteristics differ between HER2 3+ and HER2 2+/Her2 amplified cases. Virchows Archiv 2016, 469, 45–50.
[CrossRef]
16. Cronin, K.A.; Harlan, L.C.; Dodd, K.W.; Abrams, J.S.; Ballard-Barbash, R. Population-based Estimate of the Prevalence of HER-2
Positive Breast Cancer Tumors for Early Stage Patients in the US. Cancer Investig. 2010, 28, 963–968. [CrossRef] [PubMed]
17. Schettini, F.; Chic, N.; Brasó-Maristany, F.; Paré, L.; Pascual, T.; Conte, B.; Martínez-Sáez, O.; Adamo, B.; Vidal, M.; Barnadas, E.;
et al. Clinical, pathological, and PAM50 gene expression features of HER2-low breast cancer. NPJ Breast Cancer 2021, 7, 1.
[CrossRef] [PubMed]
18. Fehrenbacher, L.; Cecchini, R.S.; Geyer, C.E., Jr.; Rastogi, P.; Costantino, J.P.; Atkins, J.N.; Crown, J.P.; Polikoff, J.; Boileau, J.-F.;
Provencher, L.; et al. NSABP B-47/NRG Oncology Phase III Randomized Trial Comparing Adjuvant Chemotherapy With or
Without Trastuzumab in High-Risk Invasive Breast Cancer Negative for HER2 by FISH and With IHC 1+ or 2+. J. Clin. Oncol.
2020, 38, 444–453. [CrossRef] [PubMed]
19. Rossi, V.; Sarotto, I.; Maggiorotto, F.; Berchialla, P.; Kubatzki, F.; Tomasi, N.; Redana, S.; Martinello, R.; Valabrega, G.; Aglietta, M.;
et al. Moderate Immunohistochemical Expression of HER-2 (2+) Without HER-2 Gene Amplification Is a Negative Prognostic
Factor in Early Breast Cancer. Oncologist 2012, 17, 1418–1425. [CrossRef] [PubMed]
20. Eggemann, H.; Ignatov, T.; Bürger, E.; Kantelhardt, E.J.; Fettke, F.; Thomssen, C.; Costa, S.D.; Ignatov, A. Moderate HER2
expression as a prognostic factor in hormone receptor positive breast cancer. Endocr.-Relat. Cancer 2015, 22, 725–733. [CrossRef]
[PubMed]
21. Gilcrease, M.Z.; Woodward, W.A.; Nicolas, M.M.; Corley, L.J.; Fuller, G.N.; Esteva, F.J.; Tucker, S.L.; Buchholz, T.A. Even Low-level
HER2 Expression May be Associated with Worse Outcome in Node-positive Breast Cancer. Am. J. Surg. Pathol. 2009, 33, 759–767.
[CrossRef] [PubMed]
22. Waks, A.G.; Winer, E.P. Breast Cancer Treatment: A Review. JAMA J. Am. Med. Assoc. 2019, 321, 288–300. [CrossRef]
23. Caswell-Jin, J.L.; Plevritis, S.K.; Tian, L.; Cadham, C.J.; Xu, C.; Stout, N.K.; Sledge, G.W.; Mandelblatt, J.S.; Kurian, A.W. Change in
Survival in Metastatic Breast Cancer with Treatment Advances: Meta-Analysis and Systematic Review. JNCI Cancer Spectr. 2018,
2, pky062. [CrossRef]
24. Modi, S.; Park, H.; Murthy, R.K.; Iwata, H.; Tamura, K.; Tsurutani, J.; Moreno-Aspitia, A.; Doi, T.; Sagara, Y.; Redfern, C.; et al.
Antitumor Activity and Safety of Trastuzumab Deruxtecan in Patients With HER2-Low–Expressing Advanced Breast Cancer:
Results From a Phase Ib Study. J. Clin. Oncol. 2020, 38, 1887–1896. [CrossRef] [PubMed]
25. Banerji, U.; Van Herpen, C.M.L.; Saura, C.; Thistlethwaite, F.; Lord, S.; Moreno, V.; MacPherson, I.R.; Boni, V.; Rolfo, C.;
E De Vries, E.G.; et al. Trastuzumab duocarmazine in locally advanced and metastatic solid tumours and HER2-expressing breast
cancer: A phase 1 dose-escalation and dose-expansion study. Lancet Oncol. 2019, 20, 1124–1135. [CrossRef]
26. Pistilli, B.; Wildiers, H.; Hamilton, E.P.; Ferreira, A.A.; Dalenc, F.; Vidal, M.; Gavilá, J.; Goncalves, A.; Murias, C.;
Mouret-Reynier, M.-A.; et al. Clinical activity of MCLA-128 (zenocutuzumab) in combination with endocrine therapy
(ET) in ER+/HER2-low, non-amplified metastatic breast cancer (MBC) patients (pts) with ET-resistant disease who had
progressed on a CDK4/6 inhibitor (CDK4/6i). J. Clin. Oncol. 2020, 38, 1037. [CrossRef]
Cancers 2021, 13, 1015 16 of 18

27. Ross, J.S.; Fletcher, J.A.; Linette, G.P.; Stec, J.; Clark, E.; Ayers, M.; Symmans, W.F.; Pusztai, L.; Bloom, K.J. The HER-2/ neu Gene
and Protein in Breast Cancer 2003: Biomarker and Target of Therapy. Oncologist 2003, 8, 307–325. [CrossRef]
28. Gianni, L.; Lladó, A.; Bianchi, G.; Cortes, J.; Kellokumpu-Lehtinen, P.-L.; Cameron, D.A.; Miles, D.; Salvagni, S.; Wardley, A.;
Goeminne, J.-C.; et al. Open-Label, Phase II, Multicenter, Randomized Study of the Efficacy and Safety of Two Dose Levels of
Pertuzumab, a Human Epidermal Growth Factor Receptor 2 Dimerization Inhibitor, in Patients With Human Epidermal Growth
Factor Receptor 2–Negative Metastatic Breast Cancer. J. Clin. Oncol. 2010, 28, 1131–1137. [CrossRef] [PubMed]
29. Hickerson, A.; Clifton, G.T.; Hale, D.F.; Peace, K.M.; Holmes, J.P.; Vreeland, T.J.; Litton, J.K.; Murthy, R.K.; Lukas, J.J.;
Mittendorf, E.A.; et al. Final analysis of nelipepimut-S plus GM-CSF with trastuzumab versus trastuzumab alone to prevent
recurrences in high-risk, HER2 low-expressing breast cancer: A prospective, randomized, blinded, multicenter phase IIb trial. J.
Clin. Oncol. 2019, 37, 1. [CrossRef]
30. Filho, O.M.; Viale, G.; Trippa, L.; Li, T.; Yardley, D.A.; Mayer, I.A.; Abramson, V.G.; Arteaga, C.L.; Spring, L.; Waks, A.G.; et al.
HER2 heterogeneity as a predictor of response to neoadjuvant T-DM1 plus pertuzumab: Results from a prospective clinical trial.
J. Clin. Oncol. 2019, 37, 502. [CrossRef]
31. Ithimakin, S.; Day, K.C.; Malik, F.; Zen, Q.; Dawsey, S.J.; Bersano-Begey, T.F.; Quraishi, A.A.; Ignatoski, K.W.; Daignault, S.;
Davis, A.; et al. HER2 Drives Luminal Breast Cancer Stem Cells in the Absence of HER2 Amplification: Implications for Efficacy
of Adjuvant Trastuzumab. Cancer Res. 2013, 73, 1635–1646. [CrossRef] [PubMed]
32. Perez, E.A.; Romond, E.H.; Suman, V.J.; Jeong, J.-H.; Sledge, G.; Geyer, C.E.G., Jr.; Martino, S.; Rastogi, P.; Gralow, J.;
Swain, S.M.; et al. Trastuzumab Plus Adjuvant Chemotherapy for Human Epidermal Growth Factor Receptor 2–Positive Breast
Cancer: Planned Joint Analysis of Overall Survival From NSABP B-31 and NCCTG N9831. J. Clin. Oncol. 2014, 32, 3744–3752.
[CrossRef] [PubMed]
33. Paik, S.; Kim, C.; Wolmark, N. HER2Status and Benefit from Adjuvant Trastuzumab in Breast Cancer. N. Engl. J. Med. 2008, 358,
1409–1411. [CrossRef] [PubMed]
34. Perez, E.A.; Reinholz, M.M.; Hillman, D.W.; Tenner, K.S.; Schroeder, M.J.; Davidson, N.E.; Martino, S.; Sledge, G.W.; Harris, L.N.;
Gralow, J.R.; et al. HER2and Chromosome 17 Effect on Patient Outcome in the N9831 Adjuvant Trastuzumab Trial. J. Clin. Oncol.
2010, 28, 4307–4315. [CrossRef]
35. Burris, H.A.; Rugo, H.S.; Vukelja, S.J.; Vogel, C.L.; Borson, R.A.; Limentani, S.; Tan-Chiu, E.; Krop, I.E.; Michaelson, R.A.;
Girish, S.; et al. Phase II Study of the Antibody Drug Conjugate Trastuzumab-DM1 for the Treatment of Human Epidermal
Growth Factor Receptor 2 (HER2) –Positive Breast Cancer After Prior HER2-Directed Therapy. J. Clin. Oncol. 2011, 29, 398–405.
[CrossRef] [PubMed]
36. Krop, I.E.; Lorusso, P.; Miller, K.D.; Modi, S.; Yardley, D.; Rodriguez, G.; Guardino, E.; Lu, M.; Zheng, M.; Girish, S.; et al. A Phase
II Study of Trastuzumab Emtansine in Patients With Human Epidermal Growth Factor Receptor 2–Positive Metastatic Breast
Cancer Who Were Previously Treated With Trastuzumab, Lapatinib, an Anthracycline, a Taxane, and Capecitabine. J. Clin. Oncol.
2012, 30, 3234–3241. [CrossRef] [PubMed]
37. Gianni, L.; Colleoni, M.; Bisagni, G.; Mansutti, M.; Zamagni, C.; Del Mastro, L.; Zambelli, S.; Frassoldati, A.; Barlera, S.; Valagussa,
P.; et al. Ki67 during and after neoadjuvant trastuzumab, pertuzumab and palbociclib plus or minus fulvestrant in HER2 and
ER-positive breast cancer: The NA-PHER2 Michelangelo study. J. Clin. Oncol. 2019, 37, 527. [CrossRef]
38. Hamilton, E.; Shapiro, C.L.; Petrylak, D.; Boni, V.; Martin, M.; Del Conte, G.; Cortes, J.; Agarwal, L.; Arkenau, H.-T.; Tan, A.R.;
et al. Trastuzumab Deruxtecan (T-DXd; DS-8201) with Nivolumab in Patients with HER2-Expressing, Advanced Breast Cancer: A 2-Part,
Phase 1b, Multicenter, Open-Label Study; SABCS: San Antonio, TX, USA, 2020.
39. Eiger, D.; Pondé, N.F.; De Azambuja, E. Pertuzumab in HER2-positive early breast cancer: Current use and perspectives. Futur.
Oncol. 2019, 15, 1823–1843. [CrossRef]
40. Friess, T.S.; Bauer, A.M.B. In vivo activity of recombinant humanized monoclonal antibody 2C4 in xenografts is independent of
tumor type and degree of HER2 overexpression. Eur. J. Cancer 2002, 38, S149. [CrossRef]
41. Fisk, B.; Blevins, T.L.; Wharton, J.T.; Ioannides, C.G. Identification of an immunodominant peptide of HER-2/neu protooncogene
recognized by ovarian tumor-specific cytotoxic T lymphocyte lines. J. Exp. Med. 1995, 181, 2109–2117. [CrossRef] [PubMed]
42. Benavides, L.C.; Gates, J.D.; Carmichael, M.G.; Patel, R.; Holmes, J.P.; Hueman, M.T.; Mittendorf, E.A.; Craig, D.; Stojadinovic, A.;
Ponniah, S.; et al. The Impact of HER2/neu Expression Level on Response to the E75 Vaccine: From U.S. Military Cancer Institute
Clinical Trials Group Study I-01 and I-02. Clin. Cancer Res. 2009, 15, 2895–2904. [CrossRef] [PubMed]
43. Gall, V.A.; Philips, A.V.; Qiao, N.; Clise-Dwyer, K.; Perakis, A.A.; Zhang, M.; Clifton, G.T.; Sukhumalchandra, P.; Mao, Z.;
Reddy, S.M.; et al. Trastuzumab Increases HER2 Uptake and Cross-Presentation by Dendritic Cells. Cancer Res. 2017, 77,
5374–5383. [CrossRef] [PubMed]
44. Junttila, T.T.; Li, G.; Parsons, K.; Phillips, G.L.; Sliwkowski, M.X. Trastuzumab-DM1 (T-DM1) retains all the mechanisms of action
of trastuzumab and efficiently inhibits growth of lapatinib insensitive breast cancer. Breast Cancer Res. Treat. 2011, 128, 347–356.
[CrossRef] [PubMed]
45. Shafi, H.; Astvatsaturyan, K.; Chung, F.; Mirocha, J.; Schmidt, M.; Bose, S. Clinicopathological significance of HER2/neu genetic
heterogeneity in HER2/neu non-amplified invasive breast carcinomas and its concurrent axillary metastasis. J. Clin. Pathol. 2013,
66, 649–654. [CrossRef] [PubMed]
Cancers 2021, 13, 1015 17 of 18

46. Ogitani, Y.; Aida, T.; Hagihara, K.; Yamaguchi, J.; Ishii, C.; Harada, N.; Soma, M.; Okamoto, H.; Oitate, M.; Arakawa, S.; et al.
DS-8201a, A Novel HER2-Targeting ADC with a Novel DNA Topoisomerase I Inhibitor, Demonstrates a Promising Antitumor
Efficacy with Differentiation from T-DM1. Clin. Cancer Res. 2016, 22, 5097–5108. [CrossRef]
47. Van Der Lee, M.M.; Groothuis, P.G.; Ubink, R.; Van Der Vleuten, M.A.; Van Achterberg, T.A.; Loosveld, E.M.; Damming, D.;
Jacobs, D.C.; Rouwette, M.; Egging, D.F.; et al. The Preclinical Profile of the Duocarmycin-Based HER2-Targeting ADC SYD985
Predicts for Clinical Benefit in Low HER2-Expressing Breast Cancers. Mol. Cancer Ther. 2015, 14, 692–703. [CrossRef] [PubMed]
48. Le Joncour, V.; Martins, A.; Puhka, M.; Isola, J.; Salmikangas, M.; Laakkonen, P.; Joensuu, H.; Barok, M. A Novel Anti-HER2
Antibody–Drug Conjugate XMT-1522 for HER2-Positive Breast and Gastric Cancers Resistant to Trastuzumab Emtansine. Mol.
Cancer Ther. 2019, 18, 1721–1730. [CrossRef]
49. Geuijen, C.A.; De Nardis, C.; Maussang, D.; Rovers, E.; Gallenne, T.; Hendriks, L.J.; Visser, T.; Nijhuis, R.; Logtenberg, T.;
De Kruif, J.; et al. Unbiased Combinatorial Screening Identifies a Bispecific IgG1 that Potently Inhibits HER3 Signaling via
HER2-Guided Ligand Blockade. Cancer Cell 2018, 33, 922–936. [CrossRef] [PubMed]
50. Staudacher, A.H.; Brown, M.P. Antibody drug conjugates and bystander killing: Is antigen-dependent internalisation required?
Br. J. Cancer 2017, 117, 1736–1742. [CrossRef]
51. Ogitani, Y.; Hagihara, K.; Oitate, M.; Naito, H.; Agatsuma, T. Bystander killing effect of DS -8201a, a novel anti-human epidermal
growth factor receptor 2 antibody–drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity.
Cancer Sci. 2016, 107, 1039–1046. [CrossRef]
52. Modi, S.; Saura, C.; Yamashita, T.; Park, Y.H.; Kim, S.-B.; Tamura, K.; Andre, F.; Iwata, H.; Ito, Y.; Tsurutani, J.; et al. Trastuzumab
Deruxtecan in Previously Treated HER2-Positive Breast Cancer. N. Engl. J. Med. 2020, 382, 610–621. [CrossRef]
53. Modi, S.; Ohtani, S.; Lee, C.; Wang, Y.; Saxena, K.; Cameron, D.A. Abstract OT1-07-02: A phase 3, multicenter, randomized,
open-label trial of [fam-] trastuzumab deruxtecan (T-DXd; DS-8201a) vs investigator’s choice in HER2-low breast cancer
(DESTINY-Breast04). Ongoing Clin. Trials 2020, 80, OT1-07. [CrossRef]
54. Bardia, A.; Barrios, C.; Dent, R. Trastuzumab Deruxtecan (T-DXd; DS-8201) vs Investigator’s Choice of Chemotherapy in Patients
with Hormone Receptor-Positive (HR+), HER2 Low Metastatic Breast Cancer Whose Disease has Progressed on Endocrine Therapy in the
Metastatic Setting: A Randomized; SABCS: San Antonio, TX, USA, 2020.
55. Dokter, W.; Ubink, R.; Van Der Lee, M.; Van Der Vleuten, M.; Van Achterberg, T.; Jacobs, D.; Loosveld, E.; Dobbelsteen, D.V.D.;
Egging, D.; Mattaar, E.; et al. Preclinical Profile of the HER2-Targeting ADC SYD983/SYD985: Introduction of a New Duocarmycin-
Based Linker-Drug Platform. Mol. Cancer Ther. 2014, 13, 2618–2629. [CrossRef]
56. Hamilton, E.P.; Barve, M.A.; Bardia, A.; Beeram, M.; Bendell, J.C.; Mosher, R.; Hailman, E.; Bergstrom, D.A.; Burris, H.A.;
Soliman, H.H. Phase 1 dose escalation of XMT-1522, a novel HER2-targeting antibody-drug conjugate (ADC), in patients (pts)
with HER2-expressing breast, lung and gastric tumors. J. Clin. Oncol. 2018, 36, 2546. [CrossRef]
57. Alsina, M.; Boni, V.; Schellens, J.H.; Moreno, V.; Bol, K.; Westendorp, M.; Sirulnik, L.A.; Tabernero, J.; Calvo, E. First-in-human
phase 1/2 study of MCLA-128, a full length IgG1 bispecific antibody targeting HER2 and HER3: Final phase 1 data and
preliminary activity in HER2+ metastatic breast cancer (MBC). J. Clin. Oncol. 2017, 35, 2522. [CrossRef]
58. Bianchini, G.; Gianni, L. The immune system and response to HER2-targeted treatment in breast cancer. Lancet Oncol. 2014, 15,
e58–e68. [CrossRef]
59. Pernas, S.; Tolaney, S.M. HER2-positive breast cancer: New therapeutic frontiers and overcoming resistance. Ther. Adv. Med Oncol.
2019, 11, 1–16. [CrossRef] [PubMed]
60. Loi, S.; Giobbe-Hurder, A.; Gombos, A.; Bachelot, T.; Hui, R.; Curigliano, G.; Campone, M.; Biganzoli, L.; Bonnefoi, H.;
Jerusalem, G.; et al. Abstract GS2-06: Phase Ib/II study evaluating safety and efficacy of pembrolizumab and trastuzumab in
patients with trastuzumab-resistant HER2-positive metastatic breast cancer: Results from the PANACEA (IBCSG 45-13/BIG
4-13/KEYNOTE-014) study. Gen. Sess. Abstr. 2018, 78, GS2-06. [CrossRef]
61. Bracci, L.; Schiavoni, G.; Sistigu, A.; Belardelli, F. Immune-based mechanisms of cytotoxic chemotherapy: Implications for the
design of novel and rationale-based combined treatments against cancer. Cell Death Differ. 2014, 21, 15–25. [CrossRef]
62. Iwata, T.N.; Ishii, C.; Ishida, S.; Ogitani, Y.; Wada, T.; Agatsuma, T. A HER2-Targeting Antibody–Drug Conjugate, Trastuzumab
Deruxtecan (DS-8201a), Enhances Antitumor Immunity in a Mouse Model. Mol. Cancer Ther. 2018, 17, 1494–1503. [CrossRef]
[PubMed]
63. Collins, D.; Jacob, W.; Cejalvo, J.M.; Ceppi, M.; James, I.; Hasmann, M.; Crown, J.; Cervantes, A.; Weisser, M.; Bossenmaier, B.
Direct estrogen receptor (ER) / HER family crosstalk mediating sensitivity to lumretuzumab and pertuzumab in ER+ breast
cancer. PLoS ONE 2017, 12, e0177331. [CrossRef] [PubMed]
64. Jhaveri, K.; Hamilton, E.; Loi, S.; Schmid, P.; Darilay, A.; Gao, C.; Patel, G.; Wrona, M.; Andre, F. Trastuzumab Deruxtecan (T-DXd;
DS-8201) in Combination with Other Anticancer Agents in Patients with HER2-Low Metastatic Breast Cancer: A Phase 1b, Open-label,
Multicenter, Dose-Finding and Dose-Expansion Study (DESTINY-Breast08); SABCS: San Antonio, TX, USA, 2020.
65. O’Leary, B.; Finn, R.S.; Turner, N.C. Treating cancer with selective CDK4/6 inhibitors. Nat. Rev. Clin. Oncol. 2016, 13, 417–430.
[CrossRef] [PubMed]
66. Witkiewicz, A.K.; Cox, D.; Knudsen, E.S. CDK4/6 inhibition provides a potent adjunct to Her2-targeted therapies in preclinical
breast cancer models. Genes Cancer 2014, 5, 261–272. [CrossRef] [PubMed]
Cancers 2021, 13, 1015 18 of 18

67. Goel, S.; Wang, Q.; Watt, A.C.; Tolaney, S.M.; Dillon, D.A.; Li, W.; Ramm, S.; Palmer, A.C.; Yuzugullu, H.; Varadan, V.; et al.
Overcoming Therapeutic Resistance in HER2-Positive Breast Cancers with CDK4/6 Inhibitors. Cancer Cell 2016, 29, 255–269.
[CrossRef] [PubMed]
68. Gianni, L.; Bisagni, G.; Colleoni, M.; Del Mastro, L.; Zamagni, C.; Mansutti, M.; Zambetti, M.; Frassoldati, A.; De Fato, R.;
Valagussa, P.; et al. Neoadjuvant treatment with trastuzumab and pertuzumab plus palbociclib and fulvestrant in HER2-positive,
ER-positive breast cancer (NA-PHER2): An exploratory, open-label, phase 2 study. Lancet Oncol. 2018, 19, 249–256. [CrossRef]
69. Rinnerthaler, G.; Gampenrieder, S.P.; Greil, R. HER2 Directed Antibody-Drug-Conjugates beyond T-DM1 in Breast Cancer. Int. J.
Mol. Sci. 2019, 20, 1115. [CrossRef]
70. Diéras, V.; Harbeck, N.; Budd, G.T.; Greenson, J.K.; Guardino, A.E.; Samant, M.; Chernyukhin, N.; Smitt, M.C.; Krop, I.E.
Trastuzumab Emtansine in Human Epidermal Growth Factor Receptor 2–Positive Metastatic Breast Cancer: An Integrated Safety
Analysis. J. Clin. Oncol. 2014, 32, 2750–2757. [CrossRef] [PubMed]
71. Montemurro, F.; Ellis, P.; Anton, A.; Wuerstlein, R.; Delaloge, S.; Bonneterre, J.; Quenel-Tueux, N.; Linn, S.C.; Irahara, N.;
Donica, M.; et al. Safety of trastuzumab emtansine (T-DM1) in patients with HER2-positive advanced breast cancer: Primary
results from the KAMILLA study cohort 1. Eur. J. Cancer 2019, 109, 92–102. [CrossRef]
72. Saliba, F.; Hagipantelli, R.; Misset, J.L.; Bastian, G.; Vassal, G.; Bonnay, M.; Herait, P.; Cote, C.; Mahjoubi, M.; Mignard, D.; et al.
Pathophysiology and therapy of irinotecan-induced delayed-onset diarrhea in patients with advanced colorectal cancer: A
prospective assessment. J. Clin. Oncol. 1998, 16, 2745–2751. [CrossRef]
73. Powell, C.; Camidge; Gemma, A.; Kusumoto, M.; Baba, T.; Kuwano, K.; Bankier, A.; Kiura, K.; Tamura, K.; Modi, S.; et al. Abstract
P6-17-06: Characterization, monitoring and management of interstitial lung disease in patients with metastatic breast cancer:
Analysis of data available from multiple studies of DS-8201a, a HER2-targeted antibody drug conjugate with a topoisomera.
Poster Sess. Abstr. 2019, 79, P6-17. [CrossRef]
74. Pondé, N.F.; Lambertini, M.; De Azambuja, E. Twenty years of anti-HER2 therapy-associated cardiotoxicity. ESMO Open 2016, 1,
e000073. [CrossRef] [PubMed]
75. Lenz, H. Management and Preparedness for Infusion and Hypersensitivity Reactions. Oncologist 2007, 12, 601–609. [CrossRef]
[PubMed]